Bcl-2-modifying factor (bmf) sequences and their use in modulating apoptosis

ABSTRACT

The present invention relates generally to novel molecules capable of, inter alia, modulating apoptosis in mammalian cells and to genetic sequences encoding same. More particularly, the present invention relates to a novel member of the Bcl-2 family of proteins, referred to herein as “Bmf”, and to genetic sequences encoding same and to regulatory sequences such as a promoter sequence directing expression of Bmf. Bmf comprises a BH3 domain which facilitates interaction to pro-survival Bcl-2 family members thereby triggering apoptosis. Bmf is regarded, therefore, as a BH3-only molecule. The molecules of the present invention are useful, for example, in therapy, diagnosis, antibody generation and as a screening tool for therapeutic agents capable of modulating physiological cell death or survival and/or modulating cell cycle entry. The present invention further contemplates genetically modified animals in which one or both alleles of Bmf are mutated or partially or wholly deleted alone or in combination with a mutation in one or both alleles of another Bcl-2-type molecule such as but not limited to Bim. The genetically modified animals are useful inter alia in screening for agents which ameliorate the symptoms of diseases caused by defects in apoptosis or which specifically promote apoptosis of target cells.

FIELD OF THE INVENTION

The present invention relates generally to novel molecules capable of,inter alia, modulating apoptosis in mammalian cells and to geneticsequences encoding same. More particularly, the present inventionrelates to a novel member of the Bcl-2 family of proteins, referred toherein as “Bmf”, and to enetic sequences encoding same and to regulatorysequences such as a promoter sequence directing expression of Bmf. Bmfcomprises a BH3 domain which facilitates interaction- to pro-survivalBcl-2 family members thereby triggering apoptosis. Bmf is regarded,therefore, as a BH3-only molecule. The molecules of the presentinvention are useful, for example, in therapy, diagnosis, antibodygeneration and as a screening tool for therapeutic agents capable ofmodulating physiological cell death or survival and/or modulating cellcycle entry. The present invention further contemplates geneticallymodified animals in which one or both alleles of Bmf are mutated orpartially or wholly deleted alone or in combination with a mutation inone or both alleles of another Bcl-2-type molecule such as but notlimited to Bim. The genetically modified animals are useful inter aliain screening for agents which ameliorate the symptoms of diseases causedby defects in apoptosis or which specifically promote apoptosis oftarget cells.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Apoptosis, the physiologic and genetically modulated process of celldeath, is of central importance for modelling tissues and maintaininghomeostasis in multicellular organisms (Kerr et al., Br. J. Cancer 26:239-257, 1972; Jacobson et al., Cell 88: 347-354, 1997). Great progressis being made towards understanding the biochemistry underlying thisintrinsic suicide program. The cellular apoptotic effector moleculesinclude a set of cysteine proteinases, termed caspases, that degradecritical cellular substrates (Nicholson et al., Trends Biochem. Sci. 22:299-306, 1997). The regulatory machinery that governs the activation ofthe caspases is less well understood. However, a family of proteins ofwhich Bcl-2 is the prototypic molecule (and is referred to as the Bcl-2family of proteins) plays a central role (Jacobson, Curr. Biol. 7:R277-R281, 1997; Reed, Nature 387: 773-776, 1997; Kroemer, Nature Med.3: 614-620, 1997; Adams and Cory, Science 281: 1322-1326, 1998).

Bcl-2 was the first intracellular regulator of apoptosis to beidentified (Vaux et al., Nature 335: 440-442, 1988) and high levelsenhance cell survival under diverse cytotoxic conditions. Other cellularhomologs, such as BCl-x_(L) (Boise et al., Cell 74: 597-608, 1993) andBcl-w (Gibson et al., Oncogene 13: 665675, 1996), also enhance cellsurvival, as do more distantly related viral homologs, such as theadenovirus E1B 19K protein (White et al., Mol. Cell. Biol. 12:2570-2580, 1992) and Epstein-Barr virus BHRF-1 (Henderson er al., ProcNatl. Acad. Sci. USA 90: 8479-8483, 1993).

Pro-apoptotic BH3-only members of the Bcl-2 family are essential forinitiation of apoptosis in species as distantly related as mice and C.elegans (Huang and Strasser, Cell 103: 839, 2000). EGL-1, the so faronly recognized BH3-only protein in C. elegans, is required for alldevelopmentally programmed cell deaths in this organism. In contrast, anumber of BH3-only proteins have already been identified in mammals:Blk, Bad, Bik, Hrk, Bid, Bim, Noxa and Puma. Experiments with knock-outmice have shown that different apoptotic stimuli require distinctBH3-only proteins for their initiation. (Huang and Strasser, 2000,supra). For example, Bim is essential for apoptosis induced by cytokinewithdrawal or antigen receptor stimulation, but is dispensable for celldeath induced by glucocorticoids (Bouillet et al., Science 286: 1735,1999; Bouillet et al., Nature 415, 922, 2002). In contrast, Bid isinvolved in Fas-induced killing of hepatocytes (Yin et al., Nature 400:886, 1999). Moreover, different cell types may require distinct BH3-onlyproteins for their developmentally programmed death. Consistent withthis idea, Bim-deficient mice have an abnormal accumulation of lymphoidand myeloid cells but erythropoiesis appears normal (Bouillet et al.,1999, supra). These results indicate that individual mammalian BH3-onlyproteins have specific functions.

The pro-apoptotic activity of BH3-only proteins is subject to stringentcontrol. In C. elegans, EGL-1 is regulated by the transcriptionalrepresser TRA-1A in a group of neurons that is required for egg-laying(Conradt and Horvitz, Cell 93: 519, 1998). Some mammalian BH3-onlyproteins are also subject to transcriptional regulation. For example,Noxa was discovered as a p53-inducible gene and is therefore a primecandidate for mediating DNA damage-induced apoptosis (Oda et al.,Science 288: 1053, 2000). Several mammalian BH3-only proteins can alsobe regulated post-translationally (Huang and Strasser, 2000, supra). Ingrowth factor-stimulated cells, Bad is phosphorylated and sequesteredaway from pro-survival Bcl-2 family members by binding to 14-3-3scaffold proteins (Zha et al, Cell 87: 619, 1996). In healthy cells, Bimis sequestered to the microtubular dynein motor complex by binding todynein light chain, DLC1/LC8 (Puthalakath et al., Mol. Cell 3: 287,1999). Certain apoptotic stimuli, such as UV-radiation or treatment withtaxol, free Bim (still bound to DLC1) and allow it to translocate to,bind and inactivate pro-survival Bcl-2 family members. This processoccurs independently of the cell death executioner cysteine proteases(caspases) and therefore constitutes an upstream signalling event inapoptosis (Puthalakath et al., 1999, supra). In contrast, thepro-apoptotic activity of Bid is unleashed upon cleavage by a variety ofcaspases (e.g. caspase-8) or by the serine protease granzyme B (Li etal., Cell 94: 491-501, 1998; Luo et al., Cell 94: 481-490, 1998),indicating that it functions as part of an amplification mechanismrather than as an initiator of apoptosis. These observations demonstratethat through sequestration to specific sites in the cell, differentBH3-only proteins function as sensors for distinct forms ofintracellular stress.

In work leading to the present invention, the inventors sought novelBH3-only proteins which played a role in embryogenesis. In accordancewith the present invention, the inventors cloned “Bmf” (Bcl-2 modifyingfactor) which was identified through yeast 2-hybrid screening of a day17 mouse embryonic library using Mcl-1 as bait. Bmf is proposed toinduce cell death and act as a “death-ligand” for certain or all membersof the pro-survival Bcl-2 family. The identification of this new genepermits the identification and rational design of a range of productsfor use in therapy, diagnosis, antibody generation and involvingmodulation of physiological cell death. These therapeutic molecules mayact as either, antagonists or agonists of Bmf's friction and will beuseful in cancer, autoimmune or degenerative disease therapy.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A sequence listing is provided after the claims.

Specific mutations in an amino acid sequence are represented herein as“X₁nX₂” where X₁ is the original amino acid residue before mutation, nis the residue number and X₂ is the mutant amino acid. Reference to Xnis a reference to a particular amino acid in an amino acid sequencewhere X is the amino acid and n is the residue number. The abbreviationX may be to the three letter or single letter amino acid code.

The present invention is predicated in part on the identification of anovel member of the pro-survival Bcl-2 family. This protein is referredto herein as “Bcl-2 modifying factor” or “Bmf”. The protein wasidentified by yeast 2-hybrid screening of a mouse embryonic libraryusing Mcl-1 as bait. Bmf is an apoptosis-inducing BH3-only protein andis activated by anoikis.

Accordingly, one aspect of the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingone or more of the identifying characteristics of Bmf or a derivative orhomolog thereof.

Another aspect of the present invention provides a nucleic acid moleculecomprising a nucleotide sequence encoding or complementary to a sequenceencoding an no acid sequence substantially as set forth in one of SEQ IDNO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative orhomolog thereof or having at least about 45% or greater similarity toone or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8or a derivative or homolog thereof.

Yet another aspect of the present invention contemplates a nucleic acidmolecule comprising a nucleotide sequence substantially as set forth inone of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or aderivative or homolog thereof capable of hybridising to one of SEQ IDNO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 under low stringencyconditions and which encodes an amino acid sequence corresponding to anamino acid sequence set forth in one of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:6 or SEQ ID NO:8 or a sequence having at least about 45%similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6or 8.

Still yet another aspect of the present invention contemplates a nucleicacid molecule comprising a sequence of nucleotides substantially as setforth in SEQ ID NOS:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

Still another aspect of the present invention is directed to an isolatednucleic acid molecule encoding bmf or a derivative thereof, said nucleicacid molecule selected from the list consisting of:—

-   (i) a nucleic acid molecule comprising a nucleotide sequence    encoding the amino acid sequence set forth in one of SEQ ID NO:2 or    SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog    thereof or having at least about 45% similarity to one or more of    SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8;-   (ii) a nucleic acid molecule comprising a nucleotide sequence    substantially as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or    SEQ ID NO:5 or SEQ ID NO:7 or a derivative or homolog thereof;-   (iii) a nucleic acid molecule capable of hybridizing under low    stringency conditions to the nucleotide sequence substantially as    set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ    ID NO:7 a derivative or homolog and encoding an amino acid sequence    corresponding to an amino acid sequence as set forth in one of SEQ    ID NO:SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 a    derivative or homolog, or a sequence having at least about 45%    similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID    NO:6 or SEQ ID NO:8;-   (iv) a nucleic acid molecule capable of hybridizing to the nucleic    acid molecule of paragraphs (i) or (ii) or (iii) under low    stringency conditions and encoding an amino acid sequence having at    least about 45% similarity to one or more of SEQ ID NO:2 or SEQ ID    NO:4 or SEQ ID NO:6 or SEQ ID NO:8; and-   (v) a derivative or mammalian homolog of the nucleic acid molecule    of paragraphs (i) or (ii) or (iii) or (iv).

A further aspect of the present invention is directed to an isolatedpolypeptide selected from the list consisting of:—

-   (i) a polypeptide having an amino acid sequence substantially as set    forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID    NO:8 or derivative or homolog thereof or a sequence having at least    about 45% similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or    SEQ ID NO:6 or SEQ ID NO:8;-   (ii) a polypeptide encoded by a nucleotide sequence substantially as    set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ    ID NO:7 or derivative or homolog thereof or a sequence encoding an    amino acid sequence having at least about 45% similarity to one or    more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8;-   (iii) a polypeptide encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in one of SEQ ID    NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or derivative or    homolog thereof under low stringency conditions and which encodes an    amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ    ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or derivative or homolog    thereof or an amino acid sequence having at least about 45%    similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID    NO:6 or SEQ ID NO:8;-   (iv) a polypeptide as defined in paragraphs (i) or (ii) or (iii) in    homodimeric form; and-   (v) a polypeptide as defined in paragraphs (i) or (ii) or (iii) in    heterodimeric form.

Yet another aspect of the present invention provides a method ofproducing a genetically modified non-human animal, said methodcomprising introducing into embryonic stem cells of an animal a geneticconstruct comprising a bmf nucleotide sequence carrying a single ormultiple nucleotide substitution, addition and/or deletion or inversionor insertion wherein there is sufficient bmf nucleotide sequences topromote homologous recombination with a bmf gene within the genomic ofsaid embryonic stem cells selecting for said homologous recombinationand selecting embryonic stem cells which carry a mutated bmf gene andthen generating a genetically modified animal from said embryonic stemcell.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a representation showing Bmf, a novel mammalian BH3-onlyprotein. (A) Predicted amino acid sequence of mouse and human Bmf. Thenine amino acids that are conserved with the dynein light chain-bindingmotif of Bim are indicated by a box marked with a single asterisk (*).The short BH3 region, identified by hidden Markov modeling (Krogh etal., J. Mol. Biol. 235: 1501, 1994), is indicated by a box marked withtwo asterisks (**). (B) Alignment of the BH3 region of Bmf with otherpro-apoptotic Bcl-2 family members. Black boxes indicate identical aminoacids and grey boxes indicate similar residues. (C) Wild-type Bmf, butnot a BH3 mutant, binds pro-survival Bcl-2 and Bcl-w.Co-immunoprecipitation experiments were carried out as previouslydescribed (Puthalakath et al., 1999, supra). Briefly, 293T cells weretransiently co-transfected with expression constructs for FLAG-taggedBcl-2 (or Bcl-w) and EE(Glu-Glu)-tagged Bmf or L138A mutant Bmf. Cellswere metabolically labeled with ³⁵S-methionine 24 hours aftertransfection and harvested after overnight culture. Volumes of celllysates with equivalent trichloroacetic acid (TCA)-precipitable ³⁵Scounts were used for immunoprecipitations with mAbs to the FLAG or EEepitope tags. (D) Interaction of endogenous Bmf with Bcl-2 in MCF-7cells. Lysates from 107 MCF-7 cells, prepared in lysis buffer containing1% v/v Triton X-100, were immunoprecipitated either with Bcl-2-100(anti-human Bcl-2) mAb or an isotype matched control mAb coupled tosepharose. Bound proteins were eluted from the beads by boiling inLaemmli buffer (non reducing), size fractionated on SDS-PAGE andtransferred onto nitrocellulose filters. Western blotting was performedwith a rat anti-Bmf mAb (9G10). The asterisk (*) indicates the lightchain of the mAb used for immunoprecipitation. (E) Wild-type Bmf, butnot a BH3 mutant, kills L929 fibroblasts. L929 fibroblasts weretransfected with empty vector, expression constructs for hygromycinresistance alone, or with wild-type Bmf, a BH3 mutant (L138A) of Bmf orBmf lacking its BH3 domain. Transfected cells were plated in mediumcontaining hygromycin and resulting drug-resistant colonies countedafter 10-14 days. Values are means (+/−SD) of three independentexperiments. (F and G) Expression of Bmf in cell lines and tissues. ForNorthern blot analysis (F), 4 μg of poly A⁺ RNA from various cell linesor from mouse embryos (embryonic day 9 to 1-day after birth) wereelectrophoresed, blotted and probed with a mouse bmf cDNA probe. Probingwith a gapdh cDNA clone was used as the loading control. For Westernblot analysis (G), 50 μg of total protein from various mouse tissues wassize-fractionated by SDS-PAGE, electroblotted onto nitrocellulosefilters and probed with affinity-purified rabbit polyclonal antibodiesto Bmf. Probing with a monoclonal antibody to HSP70 served as theloading control.

FIG. 2 is a representation showing Bmf is regulated by interaction withDLC2. (A) Expression of bmf mRNA in thymocytes treated with variousapoptotic stimuli. Total RNA was isolated from thymocytes (freshlyisolated) or at the indicated time points after culture in the absenceof cytokines or treatment with dexamethasone (1 μM), γ-radiation (10 Gy)or ionomycin (1 μg/mL). These conditions all induce substantialapoptosis and, hence, no RNA could be harvested after 7 hours oftreatment. Then 2 μg RNA was reverse transcribed using AMV reversetranscriptase. Five fold dilutions of the cDNA were subjected to PCPanalysis using bmf specific primers. After transfer of the PCR products,nitrocellulose filters were probed with a ³²P-labeled internal bmfoligonucleotide probe. (B) Bmf binds to DLC2 through its dynein lightchain binding region. Co-immunoprecipitation experiments were performedas described in the legend to FIG. 1C, from lysates of 293T cellstransiently expressing FLAG-tagged DLC2 and EE-tagged wt Bmf; a B3H3mutant (L138A) of Bmf or DLC binding region mutants of Bmf (A69P orAAA), Bid or Bax. The asterisk (*) indicates the light chain of the mAbused for immunoprecipitation (C) Interaction with DLC2 regulates thepro-apoptotic potency of Bmf. FDC-P1 cells stably expressing Bcl-2 plusEE-tagged wt Bmf, a BH3 mutant (L138A) of Bmf or DLC binding regionmutants of Bmf (A69P or AAA) were deprived of IL-3 for 1-6 days. Cellviability was assessed by propidium iodide staining and flow cytometricanalysis. Values are means (+/−SD) of three independent experiments donewith four independent clones of each genotype.

FIG. 3 is a photographic representation showing that Bmf associates withthe actin-based myosin V motor complex through DLC2. (A) Lysates from10⁷ MCF-7 cells were separated into P1, P2 and S fractions. Proteinsfrom each fraction were then size-fractionated by SDS-PAGE, transferredonto nitrocellulose and probed with mAbs specific to Bmf, Bim_(L)(O'Reilly et al., Biotechniques 25: 824, 1998), myosin V (Espreafico etal., J. Cell Biol. 119: 1541, 1992) or dynein intermediate chain IC74(Sigma). (B) MCF-7 cells were treated for 3 hours with eithercytochalasin D (10 μM) or toxin B (10 ng/mL), then fractionated andprocessed as described under (A). (C) Characterization of novel mAbsthat recognize both DLC1/LC8 and DLC2, or just DLC1/LC8. Extracts from293T cells transiently expressing FLAG-tagged DLC1 or DLC2 were run onSDS-PAGE gels, electroblotted onto nitrocellulose membranes and probedwith rat monoclonal antibodies 11F7 (which recognizes both DLC1 andDLC2) or 10D6 (which recognizes only DLC1). The faint bands of lowermolecular weight marked by arrows indicate endogenous DLC1. (D) Myosin Vis associated mostly with DLC2 whereas dynein predominantly associateswith DLC1/LC8. Cytoplasmic dynein was enriched from MCF-7 cells (Paschalet al., Methods Enzymol. 196: 181, 1991) and myosin V was purified frommouse spleen (m) or chicken brain (c) (Cheney, Methods Enzymol. 298: 3,1998). These enriched fractions were analyzed by Western blotting usingrat mAbs 11F7 (recognizes DLC1/LC8 and DLC2) or 10D6 (recognizes-onlyDLC1/LCS). Nitrocellulose membranes were probed with antibodies tomyosin V or IC74 (Sigma) to demonstrate purity of the myosin and dyneinmotor fractions. (E) Extracts from mouse spleen cells (200 μg protein)were incubated for 3 hours at 4° C. with recombinant GST or GST-taggedFADD, Bmf or Bim_(L) proteins, and the bound proteins recovered onglutathione sepharose beads. Bound proteins were eluted from the beadsby boiling in Laemmli buffer (non-reducing), size-fractionated bySDS-PAGE and electro-blotted onto nitrocellulose membranes, which wereprobed with an antibody to myosin V (Espreafico et al., 1992, supra).The nitrocellulose membrane was stained with amido black (bottom panel)to document that comparable amounts of proteins were used in the pulldown experiments. (F) Lysates from 10⁷ MCF-7 cells were fractionatedthrough a 5-20% w/v sucrose gradient. The pellet and soluble fractionswere analyzed by Western blotting for the presence of Bmf, Bim, DLC1/LC8or DLC2.

FIG. 4 is a photographic representation showing that Bmf and Bim arereleased from their sequestration sites in response to distinctapoptotic stimuli. (A) MCF-7 cells were cultured in the presence of thebroad-spectrum caspase inhibitor zVAD-fmk (50 μM). Lysates from control(untreated) cells were compared with those from cells subjected tovarious apoptotic stimuli, including anoikis (culturing cells for 24hours in suspension on poly-hema coated bacterial Petri dishes),UV-irradiation (100 J/m²), paclitaxel (taxol 1 μM). Lysates of 10⁷ cellswere fractionated through sucrose gradients. The pellet and solublefractions were collected and analyzed by Western blotting for Bmf andBim using specific monoclonal antibodies. (B) During anoikis, Bmftranslocates to mitochondria and binds to Bcl-2. Mitochondria werepurified as previously described from 2×10⁸ healthy MCF-7 cells or cellssubjected to anoikis. Mitochondrial proteins were extracted in lysisbuffer containing 1% v/v Triton X-100 (Puthalakath et al., 1999, era).Immunoprecipitations were performed with anti-human Bcl-2 mAb (Bcl2-100) bound to sepharose beads. Bound proteins were eluted by boilingthe beads in Laemmli buffer (non-reducing), size-fractionated bySDS-PAGE, electroblotted onto nitrocellulose membranes and probed withmAbs to Bcl-2, Bmf or dynein light chains.

FIG. 5A is a diagrammatic representation showing the genomicorganization of the bmf gene locus of the mouse.

FIG. 5B is a diagrammatic representation of a bmf targeting construct inNFB193neo or NEB193hygro for use in generating knockout mice.

Single and three letter abbreviations used throughout the specificationare defined below. SINGLE AND THREE LETTER AMINO ACID ABBREVIATIONSTHREE-LETTER ONE-LETTER AMINO ACID ABBREVIATION SYMBOL Alanine Ala AArginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Scrine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

A summary of sequence identifiers is provided below:— SUMMARY OF SEQENCEIDENTIFIERS SEQ ID NO: DESCRIPTION 1 Nucleotide sequence of mouse bmf 2Amino acid sequence of mouse Bmf 3 Nucleotide sequence of human bmf 4Amino acid sequence of human Bmf 5 Nucleotide sequence of BH3 domain ofmouse bmf 6 Amino acid sequence of BH3 domain of mouse Bmf 7 Nucleotidesequence of BH3 domain of human bmf 8 Amino acid sequence of BH3 domainof human bmf 9 Nucleotide sequence of mouse bmf promoter 10 Nucleotidesequence of human bmf promoter 11 5′ sense primer 12 3′ antisense primer13 internal bmf primer 14 5′ sense primer 15 3′ antisense primer 16internal primer 17 predicted amino acid sequence of mouse Bmf 18predicted amino acid sequence of human Bmf 19 partial amino acidsequence of Bmf 20 partial amino acid sequence of Bim 21 partial aminoacid sequence of EGL-1 22 partial amino acid sequence of Bak 23 partialamino acid sequence of Bax 24 partial amino acid sequence of Bid 25partial amino acid sequence of Bik 26 partial amino acid sequence of Hrk27 partial amino acid sequence of Bad

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification of anovel member of the Bcl-2 family of proteins. The protein is called“Bmf” for “Bcl-2 modifying factor”. It is proposed that in healthycells, Bmf is sequestered to the actin-based myosin V motor complex bybinding to a dynein light chain and in particular dynein light chain 2(DLC2). It is further proposed that certain apoptotic stimuli, such asanoikis, release Bmf from the myosin V motor complex allowing it totranslocate and bind to Bcl-2. Consequently, Bmf functions as a sensorof intracellular damage by sequestration to motor complexes on distinctcytoskeletal structures.

Accordingly, one aspect of the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding or complementary to asequence encoding an amino acid sequence substantially as set forth inone of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or aderivative or homolog thereof or having at least about 45% or greatersimilarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6or SEQ ID NO:8 or a derivative or homolog thereof.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity”, “percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 mononmer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerised implementations ofalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science DriveMadison, Wis., USA) or by inspection and the best alignment (i.e.resulting in the highest percentage homology over the comparison window)generated by any of the various methods selected. Reference also may bemade to the BLAST family of programs as, for example, disclosed byAltschul et al. (Nucl. Acids. Res. 25: 3389, 1997). A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal (“Current Protocols in Molecular Biology”, John Wiley & Sons Inc.,1994-1998, Chapter 15).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gln, Cys and Met) Occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

Another aspect of the present invention contemplates a nucleic acidmolecule comprising a nucleotide sequence substantially as set forth inone of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or aderivative or homolog thereof capable of hybridizing to one of SEQ IDNO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 under low stringencyconditions and which encodes an amino acid sequence corresponding to anamino acid sequence set forth in one of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:6 or SEQ ID NO:8 or a sequence having at least about 45%similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6or SEQ ID NO:8.

More particularly, the present invention contemplates a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7.

Preferably, the subject nucleic acid molecules encode a polypeptidehaving the identifying characteristics of Bmf or its homologs orderivatives including functional derivatives.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962).However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs (Bonner and Lasky,Eur. J. Biochem. 46: 83, 1974). Formamide is optional in thesehybridization conditions. Accordingly, particularly preferred levels ofstringency are defined as follows: low stringency is 6×SSC buffer, 0.1%w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/vSDS at a temperature in the range 20° C. to 65° C.; high stringency is0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.

The nucleic acid molecule according to this aspect of the presentinvention corresponds herein to “bmf”. This gene has been determined inaccordance with the present invention to induce apoptosis. The productof the bmf gene is referred to herein as “Bmf” without limiting thisinvention in any way, human bmf has been mapped to human chromosomelocation 15q14. Bmf is known as a “BH3-only” protein since the onlyBcl-2 homology region which it contains is BH3. It thereby forms a novelmember of a Bcl-2 related BH3-only pro-apoptotic group which alsocomprises, for example, Bik/Nbk, Bid, Bim and Hrk.

The nucleic acid molecule encoding bmf is preferably a sequence ofdeoxyribonucleic acids such as cDNA sequence, an mRNA sequence or agenomic sequence. A genomic sequence may also comprise exons andintrons. A genomic sequence may also include a promoter region or otherregulatory region. The bmf genetic sequence includes splice variants.

Reference hereinafter to “Bmf” and “bmf” should be understood as areference to all forms of Bmf and bmf, respectively, including, by wayof example, polypeptide and cDNA isoforms of bmf which may be identifiedas arising from alternative splicing of bmf mRNA. Reference hereinafterto Bmf and bmf in the absence of a reference to its derivatives shouldbe understood to include reference to its derivatives thereof includingany splice variants.

The protein and/or gene is preferably from a human, primate, livestockanimal (e.g. sheep, pig, cow, horse, donkey) laboratory test animal(e.g. mouse, rat, rabbit, guinea pig) companion animal (e.g. dog, cat),captive wild animal (e.g. fox, kangaroo, koala, deer), aves (e.g.chicken, geese, duck, emu, ostrich), reptile or fish.

Derivatives include fragments (such as peptides), parts, portions,chemical equivalents, mutants, homologs or mimetics from natural,synthetic or recombinant sources including fusion proteins. Derivativesmay be derived from insertion, deletion or substitution of amino acids.Amino acid insertional derivatives include amino and/or carboxylicterminal fusions as well as intrasequence insertions of single ormultiple amino acids. Insertional amino acid sequence variants are thosein which one or more amino acid residues are introduced into apredetermined site in the protein although random insertion is alsopossible with suitable screening of the resulting product. Deletionalvariants are characterized by the removal of one or more amino acidsfrom the sequence. Substitutional amino acid variants are those in whichat least one residue in the sequence has been removed and a differentresidue inserted in its place. Additions to amino acid sequencesincluding fusions with other peptides, polypeptides or proteins. Mutantsshould be understood to include, but is not limited to, the specific Bmfor bmf mutant molecules described herein. Derivatives include, forexample, peptides derived from the BH3 region, from the dynein bindingregion or from a site of phosphorylation. Peptides include, for example,molecules comprising at least 4 contiguous amino acids corresponding toat least 4 contiguous amino acids of Bmf as herein defined. Use of theterm “polypeptides” herein should be understood to encompass peptides,polypeptides and proteins.

The derivatives of Bmf include fragments having particular epitopes orparts of the entire Bmf protein fused to peptides, polypeptides or otherproteinaceous or non-proteinaceous molecules. For example, Bmf orderivative thereof may be fused to a molecule to facilitate its entryinto a cell. Analogues of Bmf contemplated herein include, but are notlimited to, modification to side chains, incorporating of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the proteinaceous molecules ortheir analogues. Derivatives of nucleic acid sequences may similarly bederived from single or multiple nucleotide substitutions, deletionsand/or additions including fusion with other nucleic acid molecules. Thederivatives of the nucleic acid molecules of the present inventioninclude oligonucleotides, PCR primers, antisense molecules, moleculessuitable for use in co-suppression and fusion of nucleic acid molecules.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-S-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and/or D-isomers of amino acids. A list of unnatural amino acid,contemplated herein is shown in Table 1. TABLE 1 Non-conventionalNon-conventional amino acid Code amino acid Code α-aminobutyric acid AbuL-N-methylalanine Nmala α-amino-α-methylbutyrate MgabuL-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagineNmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornilthineNmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmom N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

The nucleic acid molecule of the present invention is preferably inisolated form or ligated to a vector, such as an expression vector. By“isolated” is meant a nucleic acid molecule having undergone at leastone purification step and this is conveniently defined, for example, bya composition comprising at least about 10% subject nucleic acidmolecule, preferably at least about 20%, more preferably at least about30%, still more preferably at least about 40-50%, even still morepreferably at least about 60-70%, yet even still more preferably 80-90%or greater of subject nucleic acid molecule relative to other componentsas determined by molecular weight, encoding activity, nucleotidesequence, base composition or other convenient means. The nucleic acidmolecule of the present invention may also be considered, in a preferredembodiment, to be biologically pure.

In a particularly preferred embodiment, the nucleotide sequencecorresponding to bmf is a cDNA sequence comprising a sequence ofnucleotides as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ IDNO:5 or SEQ ID NO:7 or is a derivative or homolog thereof including anucleotide sequence having similarity to one of SEQ ID NO:1 or SEQ IDNO:3 or SEQ ID NO:5 or SEQ ID NO:7 and which encodes an amino acidsequence corresponding to an amino acid sequence as set forth in one ofSEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a sequencehaving at least about 45% similarity to one or more of SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8.

A derivative of the nucleic acid molecule of the present invention alsoincludes nucleic acid molecules capable of hybridizing to the nucleotidesequences as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ IDNO:5 or SEQ ID NO:7 under low stringency conditions. Preferably, saidlow stringency is at 42° C.

In another embodiment, the present invention is directed to an isolatednucleic acid molecule encoding bmf or a derivative thereof said nucleicacid molecule selected from the list consisting of:—

-   (i) a nucleic acid molecule comprising a nucleotide sequence    encoding the amino acid sequence set forth in one of SEQ ID NO:2 or    SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog    thereof or having at least about 45% similarity to one or more of    SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8;-   (ii) a nucleic acid molecule comprising a nucleotide sequence    substantially as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or    SEQ ID NO:5 or SEQ ID NO:7 or a derivative or homolog thereof;-   (iii) a nucleic acid molecule capable of hybridizing under low    stringency conditions to the nucleotide sequence substantially as    set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ    ID NO:7 a derivative or homolog and encoding an amino acid sequence    corresponding to an amino acid sequence as set forth in one of SEQ    ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative    or homolog or a sequence having at least about 45% similarity to one    or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8;-   (iv) a nucleic acid molecule capable of hybridizing to the nucleic    acid molecule of paragraphs (i) or (iii) under low stringency    conditions and encoding an amino acid sequence having at least about    45% similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ    ID NO:6 or SEQ ID NO:8; and-   (v) a derivative or mammalian homolog of the nucleic acid molecule    of paragraphs (i) or (ii) or (iii) or (iv).

Reference here to an ability to hybridize to a particular sequence (e.g.SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7) also includes,in the alternative, an ability to hybridize to its complementary form.In other words, nucleic acid molecules are encompassed which hybridizeto SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or theircomplementary forms.

The nucleic acid molecule may be ligated to an expression vector capableof expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell(e.g. yeast cells, fungal cells, insect cells, mammalian cells or plantcells). The nucleic acid molecule may be ligated or fused or otherwiseassociated with a nucleic acid molecule encoding another entity such as,for example, a signal peptide, a cytokine or other member of the Bcl-2family.

The present invention extends to the promoter for bmf from murine orother mammalian species. Nucleotide sequences comprising the murine andhuman bmf promoters are shown in SEQ ID NO:9 and SEQ ID NO:10,respectively. The present invention extends to mutants and derivativesof these promoters and their use in genetic constructs, gene therapy andin generating genetically modified animals. A mutant or derivative of apromoter includes one which comprises a nucleotide sequence having atleast 70% similarity to SEQ ID NOS:9 or 10 or which is capable ofhybridizing to SEQ ID NO:9 or SEQ ID NO:10 or their complementary formsunder low stringency conditions.

The present invention extends to the expression product of the nucleicacid molecule hereinbefore defined.

The expression product is Bmf having an amino acid sequence set forth inone of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or is aderivative or homolog thereof as defined above or is a mammalian homologhaving an amino acid sequence of at least about 45% similarity to theamino acid sequence set forth in one of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:6 or SEQ ID NO:8 or derivative or homolog thereof.

Another aspect of the present invention is directed to an isolatedpolypeptide selected from the list consisting of:—

-   (i) a polypeptide having an amino acid sequence substantially as set    forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID    NO:8 or derivative or homolog thereof or a sequence having at least    about 45% similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or    SEQ ID NO:6 or SEQ ID NO:8;-   (ii) a polypeptide encoded by a nucleotide sequence substantially as    set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ    ID NO:7 or derivative or homolog thereof or a sequence encoding an    amino acid sequence having at least about 45% similarity to one or    more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8;-   (iii) a polypeptide encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in one of SEQ ID    NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or derivative or    homolog thereof under low stringency conditions and which encodes an    amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ    ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or derivative or homolog    thereof or an amino acid sequence having at least about 45%    similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID    NO:6 or SEQ ID NO:8;-   (iv) a polypeptide as defined in paragraphs (i) or (ii) or (iii) in    homodimeric form; and-   (v) a polypeptide as defined in paragraphs (i) or (ii) or (iii) in    heterodimeric form.

As defined earlier, the present invention extends to peptides orderivatives thereof of Bmf. Preferably, said peptide comprises at leastS contiguous amino acids of the polypeptide defined in SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8. The present invention alsoextends to nucleic acid molecules encoding the peptides of the presentinvention.

Another aspect of the present invention provides a nucleic acid moleculecomprising a nucleotide sequence encoding a polypeptide having one ormore of the identifying characteristics of Bmf or a derivative orhomolog thereof.

Reference herein to “identifying characteristics” of Bmf includes one ormore of the following features:—

-   (i) a polypeptide which induces apoptosis;-   (ii) a polypeptide having an amino acid sequence substantially as    set forth in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID    NO:8 or a derivative or homolog thereof;-   (iii) a polypeptide having an amino acid sequence of at least 45%    similarity to SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID    NO:8;-   (iv) a polypeptide as defined in paragraph (ii) or (iii) which    induces apoptosis;-   (v) a polypeptide encoded by a nucleic acid sequence substantially    as set forth in SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID    NO:7 or derivative or homolog thereof;-   (vi) a polypeptide encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in one of SEQ ID    NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 under low    stringency conditions;-   (vii) a polypeptide as defined in paragraph (v) or (vi) which    induces apoptosis; and-   (viii) a non-apoptosis inducing derivative of the polypeptide    defined in paragraphs (i) to (vii).

The present invention should be understood to extend to the expressionproduct of the nucleic acid molecule according to this aspect of thepresent invention.

Although not intending to limit the invention to any one theory or modeof action, the BH3 region is responsible for some of the cytotoxicactions of Bmf. The BH3 region forms an amphipathic α-helix thatinteracts with the elongated hydrophobic cleft formed by the BH1, BH2and BH3 regions of pro-survival molecules such as, for example,BCl-x_(L). The pro-apoptotic action of Bmf reflects its ability to bindto the anti-apoptotic members of the Bcl-2 family.

Still without limiting the invention to any one theory or mode ofaction, the pro-apoptotic activity of Bmf is thought to be regulatedboth at the transcriptional level and at the post-translational level.Sequence analysis of the non-coding 5′ region of Bmf has revealed anumber of putative binding sites for transcription factors such as AP1.Bmf is proposed to interact via a dynein light chain such as DLC2. Adynein light chain is a highly conserved protein which is a component ofthe myosin V motor complex.

The interaction of Bmf with the myosin V motor complex regulates thepro-apoptotic activity of Bmf. Single or multiple amino acid mutationsin Bmf which abolish binding to the dynein light chain are encompassedby the present invention.

Accordingly, a related aspect of the present invention is directed to avariant of an isolated bmf nucleic acid molecule comprising one or morenucleotide mutations in said nucleic acid molecule resulting in at leastone amino acid addition, substitution and/or deletion to the polypeptideencoded by said variant wherein said polypeptide cannot bind, couple orotherwise associate with a dynein light chain, such as DLC2.

Preferably, the mutation results in an altered amino acid sequence inthe region which binds the dynein light chain. The present inventionshould be understood to extend to variants of Bmf comprising a mutationresulting in an amino acid addition, substitution and/or deletion in aregion functionally equivalent to the regions hereinbefore defined.

Accordingly, the present invention is more particularly directed to avariant of an isolated bmf nucleic acid molecule comprising one or morenucleotide mutations in said nucleic acid molecule resulting in at leastone amino acid addition, substitution and/or deletion in the region ofthe polypeptide encoded by said variant which binds the dynein lightchain wherein said polypeptide cannot bind, couple or otherwiseassociate with a dynein light chain.

Mutations contemplated by the present invention which occur incombination with one or more mutations in another location are alsocontemplated by the present invention.

The present invention extends to the expression products of the nucleicacid molecule variants defined according to this aspect of the presentinvention.

Accordingly, the present invention is directed to a variant of anisolated Bmf polypeptide comprising at least one amino acid addition,substitution and/or deletion wherein said polypeptide cannot bind,couple or otherwise associate with the dyncin light chain.

The present invention extends to derivatives of the nucleic acidmolecules and polypeptides according to this aspect of the presentinvention. The term “derivatives” should be understood as previouslydefined.

As hereinbefore defined, reference to “Bmf” and “bmf” should beunderstood to include reference to the variant molecules definedaccording to this aspect of the present invention.

The Bmf of the present invention may be in multimeric form meaning thattwo or more molecules are associated together. Where the same Bmfmolecules are associated together, the complex is a homomultimer. Anexample of a homomultimer is a homodimer. Where at least one Bmf isassociated with at least one non-Bmf molecule, then the complex is aheteromultimer such as a heterodimer. A heteromultimer may include amolecule of another member of the Bcl-2 family or other molecule capableof modulating apoptosis. Furthermore, the present invention contemplatesfusion, or hybrids or heteromeric dimers between Bmf and other moleculessuch as Bim.

The present invention contemplates, therefore, a method for modulatingexpression of bmf in a mammal, said method comprising administering tosaid mammal a modulating effective amount of an agent for a time andunder conditions sufficient to up-regulate or down-regulate or otherwisemodulate expression of bmf. For example, bmf antisense sequences such asoligonucleotides may be introduced into a cell to enhance the ability ofthat cell to survive. Conversely, a nucleic acid molecule encoding Bmfor a derivative thereof may be introduced to decrease the survivalcapacity of any cell expressing the endogenous bmf gene. Modulation ofthe expression of bmf should be understood to extend to modulatingtranscriptional and translation events such as the splicing pattern ofBmf RNA.

Another aspect of the present invention contemplates a method ofmodulating activity of Bmf in a mammal, said method comprisingadministering to said mammal a modulating effective amount of an agentfor a time and under conditions sufficient to increase or decrease Bmfactivity.

Modulation of said activity by the administration of an agent to amammal can be achieved by one of several techniques, including but in noway limited to introducing into said mammal a proteinaceous ornon-proteinaceous molecule which:

-   (i) modulates expression of bmf;-   (ii) functions as an antagonist of Bmf; and-   (iii) functions as an agonist of Bim.

Said proteinaceous molecule may be derived from natural or recombinantsources including fusion proteins or following, for example, naturalproduct screening. Said non-proteinaceous molecule may be, for example,a nucleic acid molecule or may be derived from natural sources, such asfor example natural product screening or may be chemically synthesized.The present invention contemplates chemical analogues of Bmf capable ofacting as agonists or antagonists of Bmf. Chemical agonists may notnecessarily be derived from Bmf but may share certain conformationalsimilarities. Alternatively, chemical agonists may be specificallydesigned to mimic certain physiochemical properties of Bmf. Antagonistsmay be any compound capable of blocking, inhibiting or otherwisepreventing Bmf from carrying out its normal or pathological biologicalfunctions. Antagonists include, but are not limited to parts of Bmf orpeptides thereof monoclonal antibodies specific for Bmf or parts of 3mm, and antisense nucleic acids or oligonucleotides which preventtranscription or translation of bmf genes or mRNA in mammalian cells.Agonists of Bmf and bmf include, for example, the derivative or variantmolecules or peptides hereinbefore defined which interact withanti-apoptotic molecules such as Bcl-2, to prevent their functionalactivity thereby promoting apoptosis. Agonists may also includemolecules capable of disrupting or preventing binding of Bmf to thedynein light chain or the interaction of dynein light chain with dyneinintermediate chain.

Said proteinaceous or non-proteinaceous molecule may act either directlyor indirectly to modulate the expression of bmf or the activity of Bmf.Said molecule acts directly if it associates with Bmf or Bmf to modulatethe expression or activity of bmf or Bmf. Said molecule acts indirectlyif it associates with a molecule other than bmf or Bmf which othermolecule either directly or indirectly modulates the expression oractivity of bmf or Bmf. Accordingly, the method of the present inventionencompasses the regulation of bmf or Bmf expression or activity via theinduction of a cascade of regulatory steps which lead to the regulationof bmf or Bmf expression or activity.

Increased bmf expression or Bmf activity is useful, for example, fortreatment or prophylaxis in conditions such as cancer and deletion ofautoreactive lymphocytes in autoimmune disease. Decreased bmf expressionor Bmf activity is useful in regulating inhibition or prevention of celldeath or degeneration such as under cytotoxic conditions during, forexample, γ-irradiation and chemotherapy or during HIV/AIDS or otherviral infections, ischaemia or myocardial infarction. Since Bmf isexpressed in germ cells, modulating bmf expression or Bmf activity isuseful, for example, as a contraceptive or method of sterlisation bypreventing generation of fertile sperm.

Another aspect of the present invention contemplates a method ofmodulating apoptosis in a mammal, said method comprising administeringto said mammal an effective amount of an agent for a time and underconditions sufficient to modulate the expression of a nucleotidesequence encoding bmf.

Yet another aspect of the present invention contemplates a method ofmodulating apoptosis in a mammal, said method comprising administeringto said mammal an effective amount of an agent for a time and underconditions sufficient to modulate the activity of Bmf.

Still another aspect of the present invention contemplates a method ofmodulating apoptosis in a mammal, said method comprising administeringto said mammal an effective amount of Bmf or bmf or derivative thereof.

The Bmf, bmf or derivative thereof or agent used may also be linked to atargeting means such as a monoclonal antibody, which provides specificdelivery of the Bmf, bmf or agent to the target cells.

In a preferred embodiment of the present invention, the Bmf, bmf oragent used in the method is linked to an antibody specific for saidtarget cells to enable specific delivery to these cells.

Modulation of Bmf or bmf may be accompanied simultaneously orsequentially with the modulation of other molecules or genes such as butnot limited to bim or Bim.

Administration of the Bmf, bmf or agent, in the form of a pharmaceuticalcomposition, may be performed by any convenient means. Bmf, bmf or agentof the pharmaceutical composition are contemplated to exhibittherapeutic activity when administered in an amount which depends on theparticular case. The variation depends, for example, on the human oranimal and the Bmf, bmf or agent chosen. A broad range of doses may beapplicable. Considering a patient, for example, from about 0.01 mg toabout 10 mg of Bmf or agent may be administered per kilogram of bodyweight per day. Dosage regimes may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily, weekly, monthly or other suitable time intervals orthe dose may be proportionally reduced as indicated by the exigencies ofthe situation. The Bmf or agent may be administered in a convenientmanner such as by the oral, intravenous (where water soluble),intranasal intraperitoneal, intramuscular, subcutaneous, intradermal orsuppository routes or implanting (e.g. using slow release molecules).With particular reference to use of Bmf or agent, these peptides may beadministered in the form of pharmaceutically acceptable nontoxic salts,such as acid addition salts or metal complexes, e.g. with zinc, iron orthe like (which are considered as salts for purposes of thisapplication). Illustrative of such acid addition salts arehydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate,citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.If the active ingredient is to be administered in tablet form, thetablet may contain a binder such as tragacanth, corn starch or gelatin;a disintegrating agent, such as alginic acid; and a lubricant, such asmagnesium stearate.

A further aspect of the present invention relates to the use of theinvention in relation to mammalian disease conditions. For example, thepresent invention is particularly applicable to, but in no way limitedto, use in therapy or prophylaxis in relation to cancer, degenerativediseases, autoimmune disorders, viral infections or for germ cellregulation.

Accordingly, another aspect of the present invention relates to a methodof treating a mammal, said method comprising administering to saidmammal an effective amount of an agent for a time and under conditionssufficient to modulate the expression of bmf wherein said modulationresults in modulation of apoptosis.

In another aspect, the present invention relates to a method of treatinga mammal, said method comprising administering to said mammal aneffective amount of an agent for a time and under conditions sufficientto modulate the activity of Bmf wherein said modulation results inmodulation of apoptosis.

In another aspect, the present invention relates to a method of treatinga mammal said method comprising administering to said mammal aneffective amount of Bmf or derivative thereof for a time and underconditions sufficient to modulate apoptosis.

Yet another aspect of the present invention relates to a method oftreating a mammal, said method comprising administering to said mammalan effective amount of bmf or derivative thereof for a time and underconditions sufficient to modulate apoptosis.

In yet another aspect, the present invention relates to the use of anagent capable of modulating the expression of bmf or derivative thereofin the manufacture of a medicament for the modulation of apoptosis.

Another aspect of the present invention relates to the use of an agentcapable of modulating the expression of Bmf or derivative thereof in themanufacture of a medicament for the modulation of apoptosis.

A further aspect of the present invention relates to the use of Bmf orbmf or derivative thereof in the manufacture of a medicament for themodulation of apoptosis.

Still yet another aspect of the present invention relates to agents foruse in modulating bmf expression wherein modulating expression of saidbmf modulates apoptosis.

A further aspect of the present invention relates to agents for use inmodulating Bmf expression wherein modulating expression of said Bmfmodulates apoptosis.

Another aspect of the present invention relates to Bmf or bmf orderivative thereof for use in modulating apoptosis.

In a related aspect of the present invention, the mammal undergoingtreatment may be human or an animal in need of therapeutic ofprophylactic treatment.

In yet another further aspect, the present invention contemplates apharmaceutical composition comprising bmf, Bmf or derivative thereof oran agent capable of modulating bmf expression or Bmf activity togetherwith one or more pharmaceutically acceptable carriers and/or diluents.bmf, Bmf or said agent are referred to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of surfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

When bmf, Bmf and/or Bmf modulators are suitably protected, they may beorally administered, for example, with an inert diluent or with anassimilable edible carrier, or it may be enclosed in hard or soft shellgelatin capsule, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet or administered viabreast milk. For oral therapeutic administration, the active ingredientmay be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers and the like. Such compositions and preparations shouldcontain at least 1% by weight of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 5 to about 80% of the weight of the unit.The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention areprepared so that an oral dosage unit form contains between about 0.1 μgand 200 mg of active compound. Alternative dosage amounts include fromabout 1 μg to about 1000 mg and from about 10 μg to about 500 mg. Thesedosages may be per individual or per kg body weight. Administration maybe per hour, day, week, month or year.

The tablets, troches, pills, capsules, creams and the like may alsocontain the components as listed hereafter. A binder such as gum,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art and except insofar as any conventional media or agent isincompatible with the active ingredient, their use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of modulating bmf expression orBmf activity. The vector may, for example, be a viral vector.

Conditions requiring modulation of physiological cell death includeenhancing survival of cells utilising, for example, antisense sequencein patients with neurodegenerative diseases, myocardial infarction,muscular degenerative disease, hypoxia, ischaemia, HIV infection or forprolonging the survival of cells being transplanted for treatment ofdisease. Alternatively, the molecules of the present invention areuseful for, for example, reducing the survival capacity of tumour cellsor autoreactive lymphocytes. The antisense sequence may also be used formodifying in vitro behaviour of cells, for example, as part of aprotocol to develop novel lines from cell types having unidentifiedgrowth factor requirements; for facilitating isolation of hybridomacells producing monoclonal antibodies, as described below; and forenhancing survival of cells from primary explants while they are beinggenetically modified.

Still another aspect of the present invention is directed to animmunointeractive molecule comprising an antigen binding portion havingspecificity for Bmf or bmf or derivative thereof.

Reference to “immunointeractive molecule” should be understood as areference to any molecule comprising an antigen binding portion or aderivative of said molecule. Examples of molecules contemplated by thisaspect of the present invention include, but are not limited to,monoclonal and polyclonal antibodies (including synthetic antibodies,hybrid antibodies, humanized antibodies, catalytic antibodies) and Tcell antigen receptor binding molecules. Preferably, said immunoreactivemolecule is a monoclonal antibody.

According to this preferred embodiment, there is provided a monoclonalantibody having specificity for Bmf or bmf or derivative thereof.

Reference to a molecule “having specificity for Bmf or bmf” should beunderstood as a reference to a molecule, such as a monoclonal antibody,having specificity for any one or more epitopes of Bmf or bmf. Theseepitopes may be conformational epitopes, linear epitopes or acombination of conformational and linear epitopes of either the nativeBmf or bmf molecule or the denatured molecule.

More preferably, there is provided a monoclonal antibody havingspecificity for Bmf.

The immunointeractive molecules of the present invention may benaturally occurring, synthetic or recombinantly produced. For example,monoclonal or polyclonal antibodies may be selected from naturallyoccurring antibodies to Bmf or bmf or may be specifically raised to Bmfor bmf. In the case of the latter, Bmf or bmf may first need to beassociated with a carrier molecule. The antibodies and/or recombinantBmf of the present invention are particularly useful as therapeutic ordiagnostic agents. Alternatively, fragments of antibodies may be usedsuch as Fab fragments. Furthermore, the present invention extends torecombinant and synthetic antibodies, to antibody hybrids and toantibodies raised against non-Bmf antigens but which are cross-reactivewith any one or more Bmf epitopes. A “synthetic antibody” is consideredherein to include fragments and hybrids of antibodies. The antibodies ofthis aspect of the present invention are particularly useful forimmunotherapy and may also be used as a diagnostic tool for assessingapoptosis or monitoring the program of a therapeutic regime.

For example, Bmf and bmf can be used to screen for naturally occurringantibodies to Bmf and bmf, respectively. These may occur, for example insome degenerative disorders.

For example, specific antibodies can be used to screen for Bmf proteins.The latter would be important, for example, as a means for screening forlevels of Bmf in a cell extract or other biological fluid or purifyingBmf made by recombinant means from culture supernatant fluid. Techniquesfor the assays contemplated herein are known in the art and include, forexample, sandwich assays, ELISA and flow cytometry.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies) directedto the first mentioned antibodies discussed above. Both the first andsecond antibodies may be used in detection assays or a first antibodymay be used with a commercially available anti-immunoglobulin antibody.An antibody as contemplated herein includes any antibody specific to anyregion of Bmf.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the protein or peptide derivatives and either type is utilizablefor immunoassays. The methods of obtaining both types of sera are wellknown in the art. Polyclonal sera are less preferred but are relativelyeasily prepared by injection of a suitable laboratory animal with aneffective amount of Bmf, or antigenic parts thereof, collecting serumfrom the animal, and isolating specific sera by any of the knownimmunoadsorbent techniques. Although antibodies produced by this methodare utilizable in virtually any type of immunoassay, they are generallyless favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example, Douillard and Hogan, Basic Facts aboutHybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein,European Journal of Immunology 6: 511-519, 1976).

Screening for immunointeractive molecules, such as antibodies, can be atime consuming and labour intensive process. However, the inventors havedeveloped a rapid and efficient flow cytometric screening procedure forthe identification of immunointereactive molecules, and in particularantibodies, directed to low abundance cytoplasmic proteins such as, butnot limited to, Bmf.

The method according to this aspect of the present invention is based onthe analysis of a population of cells, following the incubation of thesecells with the antibody of interest together with or separately to areporter molecule, said population of cells comprising both cellsexpressing the protein of interest and cells which do not express theprotein of interest. This analysis is preferably flow cytometricanalysis and the cells expressing the protein of interest are preferablytransfected with a nucleic acid molecule encoding the protein ofinterest to thereby express high levels of said protein. Where theprotein is a cytoplasmic protein the cells are permeabalized prior toincubation with the antibody of interest. By screening a population ofcells comprising both cells which do not express and cells which doexpress the protein of interest, determination of which antibodies bindto the protein of interest is simplified since where the subjectantibody is directed to the protein of interest, a double fluorescencepeak is observed. The lower intensity peak represents backgroundstaining while the higher fluorescence intensity peak is the result ofspecific staining. Where the antibody being screened according to thismethod is not directed to the protein of interest, a single peak of lowfluorescence intensity is observed. Antibodies not specific to theprotein of interest but bound to some unknown epitope present in bothpopulations of cells produces a single peak with high fluorscenceintensity. This technique provides a rapid and accurate method ofscreening for immunointeractive molecules directed to low abundanceintracytoplasmic molecules (O'Reilly, 1998, supra).

Accordingly, another aspect of the present invention provides a methodof detecting an immunointeractive molecule, in a sample, specific for aprotein of interest produced by a cell, said method comprising,contacting the sample to be tested with a population of cells comprisinga defined ratio of cells producing the protein of interest and cells notproducing the protein of interest for a time and under conditionssufficient for immunointeractive molecules, if present in said sample,to interact with said protein of interest and the subjecting saidimmunointeractive molecule-protein complex to detecting means.

Preferably said immunointeractive molecule is an antibody.

More preferably, said detecting means comprises an anti-immunoglobulinantibody labelled with a reporter molecule capable of giving adetectable signal. Even more preferably said reporter molecule isfluorochrome.

Reference to “sample” should be understood as a reference to any samplepotentially comprising an immunointeractive molecule, such as anantibody. Said immunointeractive molecule may be produced by natural,recombinant or synthetic means.

The method of the present invention is predicated on subjecting thecells incubated with the sample of the present invention to flowcytometric analysis to produce a fluorescent signal wherein adifferential fluorescent signal is indicative of antibody binding to thetarget protein expressed by said cells.

The method exemplified herein is directed, but not limited to, screeningfor immunointeractive molecules comprising an antigen binding sitedirected to epitopes of Bmf. The promyelomoncytic cell line FDC-P1 istransfected with a Bcl-2 expression construct and an EE (Glu-Glu)epitope-tagged Bmf construct. A 1:1 ratio of Bcl-2 transfected cells toBmf transfected cells are fixed, permeabilized and contacted with theimmunointeractive molecule of interest, such as a hybridoma supernatant.Visualization of antibodies bound intracellular molecules can beachieved via a number of techniques known to those skilled in the art,including, for example, the use of fluorescently labelled reportermolecules. Where the antibody of interest is directed to Bmf, a doublefluorescence peak is observed, the lower intensity peak representingbackground staining of the Bcl-2 transfected negative control cells.

In another aspect of the present invention, the molecules of the presentinvention are also useful as screening targets for use in applicationssuch as the diagnosis of disorders which are regulated by Bmf. Forexample, screening for the levels of Bmf or bmf in tissue as anindicator of a predisposition to, or the development or, cancer, adegenerative disease or infertility. The screening of this aspect of thepresent invention may also be directed to detecting mutations in Bmf orbmf.

Accordingly, another aspect of the present invention contemplates amethod for detecting Bmf in a biological sample from a subject, saidmethod comprising contacting said biological sample with animmunointeractive molecule as hereinbefore defined specific for Bmf orits derivatives thereof for a time and under conditions sufficient foran immunointeractive molecule-Bmf complex to form, and then detectingsaid complex.

Preferably said immunointeractive molecule is an antibody. Even morepreferably, said antibody is a monoclonal antibody.

Reference to biological sample according to this aspect of the presentinvention should be understood as a reference to any sample comprisingtissue from a subject, said “tissue” should be understood in itsbroadest sense to include biological fluid, biopsy samples or any otherform of tissue or fluid or extracts therefrom such as DNA or RNAproperties.

Still another aspect of the present invention contemplates a method fordetecting bmf in a biological sample from a subject, said methodcomprising contacting said biological sample with an immunointeractivemolecule as hereinbefore defined specific for bmf or its derivativesthereof for a time and under conditions sufficient for animmunointeractive molecule-bmf complex to form, and then detecting saidcomplex.

Reference to an “immunointeractive” molecule should be understood as areference to any molecule which couples, binds or otherwise associateswith bmf or Bmf or derivative thereof. For example, said interactivemolecule may be a nucleic acid molecule or an anti-nuclear antibody.

The presence of Bmf may be determined in a number of ways such as byWestern blotting, ELISA or flow cytometry procedures. Bmf mRNA or DNAmay be detected, for example, by in situ hybridization or Northernblotting or Southern blotting. These, of course, include bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favoured for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen complex, a second antibodyspecific to the antigen, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex ofantibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent. Inaccordance with the present invention the sample is one which mightcontain Bmf including cell extract, tissue biopsy or possibly serum,saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.The sample is, therefore, generally a biological sample comprisingbiological fluid but also extends to fermentation fluid and supernatantfluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody havingspecificity for the Bmf or antigenic parts thereof is either covalentlyor passively bound to a solid surface. The solid surface is typicallyglass or a polymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes or overnight if more convenient) and under suitableconditions (e.g. from room temperature to about 40° C. such as 25° C.)to allow binding of any subunit present in the antibody. Following theincubation period, the antibody subunit solid phase is washed and driedand incubated with a second antibody specific for a portion of thehapten. The second antibody is linked to a reporter molecule which isused to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, β-galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody hapten complex, allowed to bind,and then the excess reagent is washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of hapten which was present in the sample.“Reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect bmf or its derivatives.

The present invention further provides genetically modified animals inwhich one or both alleles of bmf are mutated alone or in combinationwith another mutation in one or both alleles for another Bcl-2 moleculesuch as but not limited to genes encoding Blk, Bad, Bik, Hrk, Bid, Bim,Noxa, blx3 and/or Puma. The animals may also have mutations in othergenes or alleles of genes. Preferably, the genetically modified annalsare laboratory test animals such as murine species (e.g. mice, rats),rabbits, guinea pigs or hamsters, livestock animals such as sheep, pigs,horses or cows or non-human mammals such as primates. Conveniently, andpreferably, the genetically modified animal is a murine species such asa mouse or rat.

The genetic modification is generally in the form of a mutation such asa single or multiple nucleotide substitution, deletion and/or additionor inversion or insertion. Generally, such a genetically modified animalis referred to as a “knockout” animal.

Genetically modified animals and in particular knock-out murine animalsmay be prepared by any number of means. In one method, a targeting DNAconstruct is prepared comprising a nucleotide sequence which issufficiently homologous to a target sequence such a bmf or bim to permithomologous recombination. The bmf or bim targeting sequence may beisogenic or non-isogenic to the target Bmf or Bim sequence. Thetargeting DNA construct generally comprises a selectable marker withinthe targeting sequence such that by homologous recombination, the targetbmf or bim gene is disrupted by an insertional mutation. The targetingDNA construct is generally introduced into an embryonic stem cell orembryonic stem cell line. One suitable targeting vector is shown in FIG.5A.

As an alternative to using a selectable marker, a mutation may beintroduced which induces a phenotypic change which may then be selectedor detected.

Accordingly, another aspect of the present invention provides a methodof producing a genetically modified non-human animal, said methodcomprising introducing into embryonic stem cells of an animal a geneticconstruct comprising a bmf nucleotide sequence carrying a single ormultiple nucleotide substitution, addition and/or deletion or inversionor insertion wherein there is sufficient bmf nucleotide sequences topromote homologous recombination with a bmf gene within the genome ofsaid embryonic stem cells selecting for said homologous recombinationand selecting embryonic stem cells which carry a mutated bmf gene andthen generating a genetically modified animal from said embryonic stemcell.

Preferably, the genetically modified animal is a murine species such asa mouse or rat.

The Bmf nucleotide sequence may be isogenic or non-isogenic to the bmfgene in the embryonic stem cell.

The term “isogenic” means that the bmf nucleotide sequence in theconstruct is derived from the same animal strain from which theembryonic stein cell has been derived.

The present invention further contemplates non-homologous-mediatedintegration of the target DNA sequence.

A range of selectable markers may be employed and reference may be madeto U.S. Pat. No. 5,789,215 for general methodologies.

The above method may be similarly adopted for introducing a plurality ofmutations into different genes such as, in addition to bmf, other Bcl-2genes (e.g. those encoding Bim, Blk, Bad, Bid, Hrk, Noxa or Puma) and/orother structural or regulatory genes.

Breeding protocols may also be adopted to introduce mutations or othergenetic modifications into Bmf. In one approach, an EMS or othermutagenized mouse is crossed with a non-mutagenized mouse to produce aG1 generation. The G1 generation may then be crossed with an indexstrain to produce GIFI kindreds which are then screened phenotypicallyfor mutation in bmf. Mutations in bmf may be dominant or recessive andmutations may be detected directly on bmf or by its effect on anothergene or on its effect in alleviating the effects of a first mutation onanother gene.

All genetically modified animals including knock-out mice carryingmutations in one or both bmf alleles alone or in combination withmutations in other genes such as other Bcl-2 family genes areencompassed by the present invention.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Identification and Cloning of Bmf

The inventors sought novel BH3-only proteins that played a role inembryogenesis. Since Mcl-1-deficient mice have the most severedevelopmental defect of all knock-out mice lacking pro-survival Bcl-2family members, Mcl-1 was used as bait. Bmf (Bcl-2 modifying factor) wasidentified through yeast 2-hybrid screening of a day 17 mouse embryoniclibrary using Mcl-1 as bait. The method used is as follows.

The cDNA libraries from day 17 mouse embryos or from mouse embryos fromembryonic day 9 to one day post-partum were prepared in pAD-GAL4-2.1(HybriZAP-2.1 kit, Stratagene). The bait vector was made by cloningmouse mcl-1 lacking the sequences encoding its hydrophobic C-terminusinto pGBT-9 (Clontech). Yeast transformation and plasmid rescue wereperformed as previously described (Puthalakath et al., 1999, supra).7×10⁵ clones were screened and one positive clone was obtained.Interaction between Mcl-1 and the novel protein was confirmed byβ-galactosidase staining (Puthalakath et al., 1999, supra). Sequenceanalysis revealed that the clone was a partial one lacking the 5′ end.This partial clone was used as the probe to isolate full-length clonesby screening a cDNA library derived from the p53^(−/−) KO52DA20 thymomacell line (Strasser et al., Cell 79: 329, 1994). Human bmf was isolatedby screening a human activated T cell cDNA library using mouse bmf asprobe. To screen for Bmf-interacting proteins, mouse bmf was subclonedinto a pGBT-9 derivative harboring the gene for chloramphenicolacetyltransferase as the selection marker. Out of 5×10⁶ clones screened,60 positive clones were initially selected, of which 6 were later foundto be false positives.

Detailed sequence analysis (Krogh et al., 1994, supra) revealed that Bmfharbors a BH3 domain most similar to that found in Bim, Bik and EGL-1(FIGS. 1A and B). In the yeast 2-hybrid system, Bmf interacted withMcl-1 and other pro-survival Bcl-2 proteins (Bcl-2, Bcl-x_(L) and Bcl-w)but not with the pro-apoptotic family members tested (Bax, Bid and Bad).When transiently overexpressed in 293T cells, Bmf could beco-immunoprecipitated with pro-survival Bcl-2 family members Bcl-2 andBcl-w (FIG. 1C), as well as BCl-x_(L) and Mcl-1, but did not bindpro-apoptotic Bax or the BH3-only protein Bim. The interaction of Bmfwith Bcl-2 or Bcl-w was greatly diminished by mutating the invariantleucine (L13SA) within its BH3 domain (FIG. 1C). Furthermore, mutationsof conserved residues within the BH1 (G145B) or BH2 (W188A) domain ofBcl-2, which abolish its binding to Bim (O'Connor et al., EMBO J. 17:384, 1998) or Bax (Yin et al., Nature 369: 321, 1994), also disrupt itsbinding to Bmf. Significantly, endogenous Bmf could beco-immunoprecipitated with endogenous Bcl-2 from detergent lysed MCF-7human breast carcinoma cells (FIG. 1D), excluding the possibility thatthese proteins associate only when overexpressed.

The biological activity of Bmf was investigated by transientlyoverexpressing it in Jurkat human T lymphoma cells, as well as in stablytransfected L929 mouse fibroblasts (FIG. 1E) or in IL-3-dependent FDC-P1mouse promyelocytic cells (FIG. 2C). Expression of Bmf triggeredapoptosis in 80% of Jurkat cells within 24 hours and reduced formationof L929 fibroblast colonies by about 65% (FIG. 1E). Bmf-inducedapoptosis in Jurkat cells could be blocked by the caspase inhibitorbaculovirus p35, or by co-expression of Bcl-2 or its homologs(Bcl-x_(L), Bcl-w, Mcl-1) but not by BH1 (G145E) or BH2 (W188A) domainmutants of Bcl-2. Consistent with its pro-apoptotic activity, highlevels of Bmf could be expressed stably in FDC-P1 cells only when Bcl-2(or one of its homologs) was also expressed. Such Bmf/Bcl-2co-expressing FDC-P1 cells died more rapidly than Bcl-2 expressing cellsin response to cytokine withdrawal (FIG. 2C), γ-irradiation or treatmentwith etoposide. In all the cell death assays performed, Bmf mutants thatlack the BH3 domain or have the L138A mutation in it were inert (FIGS.1E and 2C). These results establish that Bmf is a BH3-only protein thatbinds pro-survival Bcl-2 family members to initiate apoptosis.

EXAMPLE 2 Expression Patterns of Bmf

The expression pattern of Bmf was investigated by Northern blotting,RT-PCR and Western blotting. bmf mRNA was found in many cell lines of Band T lymphoid, myeloid or fibroblastoid origin and in mouse embryos atall developmental stages from E9 to birth (FIG. 1F). Western blotting ofcell lysates using affinity purified rabbit polyclonal antibodies or ratmonoclonal antibodies (described below) detected a single bandcorresponding to Bmf in many organs, with prominent levels found inpancreas, liver, kidney and hematopoietic tissues (FIG. 1G). Thus, Bmfis expressed during embryogenesis and in many adult tissues.

Monoclonal rat antibodies to dynein light chains and Bmf were generatedusing a previously published protocol (O'Reilly et al., 1998, supra). Inbrief, Wistar rats were immunized with purified recombinant mouseDLC1/LCS or mouse Bmf. Spleen cells from immunized rats were fused withSp2/0 myeloma cells. The resulting hybridoma clones were screened forproduction of specific antibodies by immunofluorescent staining and flowcytometric analyses. Hybridomas were cloned twice and antibodies werepurified either on a protein-G column (Amersham Pharmacia) or on asepharose column conjugated with MAR 18.5 (monoclonal mouse anti-ratIgκ) antibodies. Monoclonal antibody 11F7 (rat IgG 2a/κ) recognizesmouse and human DLC1/LC8 and DLC2 whereas 10D6 (rat μ/κ) detects mouseand human DLC1/LC8 but not DLC2. Monoclonal antibodies 9G10 and 12E10(both rat γ2a/κ) detect endogenous mouse and human Bmf by Westernblotting and immunoprecipitation. To generate polyclonal anti-Bmfantibodies, New Zealand White rabbits were immunized with 500 μg ofrecombinant mouse Bmf. Booster immunisations were given at intervals ofthree weeks. Serum was collected after 12 days and purified over asepharose column conjugated with recombinant mouse Bmf protein.

EXAMPLE 3 Apoptotic Structure

To assess whether bmf expression was induced by apoptotic stimuli,RT-PCR analyses were performed of mRNA from thymocytes exposed tovarious forms of stress, including cytokine deprivation, γ-irradiationor treatment with dexamethasone or ionomycin (described below). None ofthese stimuli had any impact on bmf expression (FIG. 2A), prompting theinventors to investigate whether Bmf is regulated post-translationally,perhaps by interacting with other proteins. A yeast 2-hybrid screen of amouse embryo cDNA library with Bmf as bait isolated 14 independentclones of Mcl-1 and, surprisingly, more than 40 clones encoding dyneinlight chain (DLC). In a previous screen, Bim had isolated exclusivelyDLC1/LC8 (Puthalakath et al., 1999, supra). In contrast, most dyneinlight chain clones interacting with Bmf encoded the closely relatedprotein DLC2 (Naisbitt et al., J. Neurosci. 20: 4524, 2000).Co-immunoprecipitation experiments in transiently transfected 293T cellsconfirmed the interaction of Bmf with DLC2 (FIG. 2B). Sequencecomparison revealed that Bmf has, in addition to the BH3 domain, a shortregion (aa67-DKATQTLSP) that closely resembles one in Bim(aa51-DKSTQTPSP) that mediates its binding to DLC1/LC8 (FIG. 1A). Thisis the DLC-binding motif of Bmf, because mutations within it (A69P orD67K68A69:>AAA, hereafter referred to as AAA mutation) abrogated theinteraction of Bmf with DLC2 in yeast and in mammalian cells (FIG. 2B).Moreover, upon IL-3 deprivation or γ-irradiation, FDC-P1 cellsco-expressing Bcl-2 and non-DLC2-binding mutants of Bmf died much morerapidly than those co-expressing Bcl-2 and wild-type Bmf FIG. 2C). TheseBmf mutants also suppressed the formation of L929 fibroblast coloniesmore potently than wild-type Bmf. Hence, interaction with DLC2negatively regulates the pro-apoptotic activity of Bmf.

RT-PCR analysis of bmf mRNA expression was performed using the followingprimers: 5′ (sense) primer 5′CCGGATGGATCACCAGGAATG3′ [SEQ ID NO:11], 3′(antisense) primer 5′CAGAGCTGACAAAGGCACAG3′ [SEQ ID NO:12]. Detection ofthe PCR products on Southern blots was performed using the internal bmfprimer 5′CCACTTCCTGGAGAACATCA3′ [SEQ ID NO:13]. For analysis of GAPDHexpression, the following primers were used: 5′ (sense) primer5′TGATGACATCAAGAAGGTGGTGAAG3′ [SEQ ID NO:14], 3′ (antisense) primer5′TCCTTGGAGGCCATGTAGGCCAT3′ [SEQ ID NO:15] and the internal primer5′CCCGGCATCGAAGGTGGAAGAG3′ [SEQ ID NO:16].

EXAMPLE 4 Functional Model

The question considered by the inventors is why Bmf is controlled bybinding to highly related partners, DLC-1 or DLC-2. It is proposed thatBmf is sequestered to sites within the cell in order to sense distinctstress stimuli. Separation of cellular proteins into the filamentousactin and the paclitaxel (taxol)-polymerizable microtubular fractionsrevealed that, consistent with previous results (Puthalakath et al.,1999, supra), Bim and dynein intermediate chain (IC74) largelyco-migrated with microtubular components (P2), whereas Bmf and myosin Vwere confined to the filamentous actin-containing P1 fraction (FIG. 3A).Furthermore, treating cells with actin depolymerizing agents, such ascytochalasin D or C. difficcili toxin B, released Bmf from thefilamentous actin-containing P1 fraction whereas the fractionation ofBim was unaffected (FIG. 3B).

For subcellular fractionation, 5×10⁶ MCF-7 cells were lysed in 500 μLextraction buffer containing 1% Triton-X-100. Cell debris and nucleiwere removed by centrifugation at 2000 g. The supernatant was thenincubated for 13 minutes at 37° C. with 100 μM paclitaxel (taxol) and Sunits of apyrase (Sigma). This mixture was then loaded on top of a 0.5mL cushion of 7.5% sucrose (made in the extraction buffer) andcentrifuged at 140,000 g for 30 minutes at 30° C. The pellet was savedas the microtubular P2 fraction and the supernatant as the S fraction.To obtain the actin-enriched P1 fraction without contamination bymicrotubular constituents, MCF-7 cells were cultured for 2 hrs in thepresence of 2 μg/mL colchicine and 1 μg/mL nocodazole prior to lysis.These lysates were then cleared of cell debris and nuclei (describedabove) and subsequently centrifuged for 60 minutes at 4° C. at 140,000 gto obtain the pellet (P1) fraction. For fractionation of extracts onsucrose gradients, 10⁷ cells were lysed in 500 μL extraction buffer.After removing cellular debris and nuclei, the supernatants were treatedwith 100 μM paclitaxel (taxol) plus 5 units of apyrase and incubated at37° C. for 13 minutes before loading onto a 5-20% sucrose gradient(prepared in extraction buffer containing 1% Triton X-100) andcentrifuging for 18 hours at 15° C. at 140,000 g.

The distinct localization of Bmf and Bim may be determined largely bytheir preferred dynein light chain partners. Contrary to a previousreport (Benashski et al, J. Biol. Chem. 272: 20929, 1997), by usingmonoclonal antibodies that either recognize only DLC1/LC8 or bothDLC1/LCS and DLC2 (FIG. 3C), the inventors showed that purified myosin Vmotor complexes contained DLC2 but not DLC1/LC8 (FIG. 3D). Thisobservation indicated that Bmf, by being preferentially bound to DLC2,might be complexed with myosin V on filamentous actin rather thanforming part of the dynein motor complex. Consistent with this notion,incubation of extracts from mouse spleen cells with recombinant Bmf andBim confirmed that only Bmf associated with myosin V (FIG. 3B).Furthermore, Bmf and Bim showed distinct migration patterns aftersubcellular fractionation of lysates from MCF-7 cells on sucrosegradients (FIG. 3F). Since DLC1/LC8 forms homodimers avidly and since itbinds Bim and IC74 through the same region (Lo et al., J. Biol. Chem.276: 14059, 2001), one partner of a DLC1/LC8 homodimer probablyinteracts with IC74 whilst the other binds Bim, thereby sequestering itto the microtubular dynein motor complex. It is likely that DLC2homodimers sequester Bmf to filamentous actin by binding with one arm toBmf and with the other to myosin V.

The inventors next investigated whether Bmf and Bim are activated bydistinct apoptotic stimuli using cells that express both proteinsendogenously. Consistent with our previous results (Puthalakath et al.,1999, supra), UV-irradiation of MCF-7 cells released Bim from the pelletfraction where the dynein motor complex resided. When lysates of healthyor damaged MCF-7 cells were compared by sucrose gradient centrifugation,it became apparent that Bmf also translocated from denser to lighterfractions in response to UV-irradiation (FIG. 4A). Treatment withpaclitaxel (taxol), a chemotherapeutic drug known to polymerizemicrotubules, released Bim but not Bmf (FIG. 4A). Consistent with acritical role for Bim in this pathway to apoptosis, Bim-deficientthymocytes are abnormally resistant to the cytotoxic effects ofpaclitaxel (Frisch and Ruoslahti, Science 286(5445): 1735-1738, 1999).On the other hand, anoikis (absence of cell attachment and integrinsignaling), an apoptotic stimulus that affects the actin cytoskeleton(Frisch and Ruoslahti, 1997, supra), resulted in the selective releaseof Bmf but not Bim (FIG. 4A). Since these experiments were conducted inthe presence of the broad-spectrum caspase inhibitor zVAD-fmk at aconcentration (50 μM) sufficient to block caspase activation, therelease of Bmf and/or Bim are likely to represent initiating events inapoptosis signaling rather than being a consequence of apoptoticchanges. Importantly, the inventors showed that endogenous Bmf (togetherwith DLC2) released during anoikis could be co-immunoprecipitated withendogenous Bcl-2 isolated from mitochondria (FIG. 4B). In contrast,negligible Bmf was found complexed with Bcl-2 isolated from mitochondriaof healthy cells.

Collectively, the inventors' data demonstrate that Bmf and Bim representtwo pro-apoptotic BH3-only proteins that transduce distinct deathsignals caused by different forms of cell stress. They seem to representsentinels mounted on the main cytoskeletal structures to monitor thewell-being of the cell. For example, disturbance of the microtubules bypaclitaxel activates Bim but not Bmf, whereas anoikis, which affects theactin cytoskeleton, activates Bmf but not Bim. Since deregulatedexpression of anti-apoptotic Bcl-2 can promote tumorigenesis (Strasseret al., Nature 348: 331, 1990), it is possible that abnormalities inpro-apoptotic BH3-only proteins can also cause cancer. The gene forhuman bmf is located on chromosome 15q14, identified as the site of acandidate tumor suppressor gene lost in many metastastic but not primarycarcinomas (Wick et al., Oncogene 12: 973, 1996). Anoikis has beenimplicated as a barrier against metastatic tumor growth (Ruoslahti andReed, Cell 77: 477, 1994). Metastatic tumors harboring 15q14 mutationsmay, therefore, have abnormalities in their expression or function ofBmf.

EXAMPLE 5 Generation of Bmf Knock-Out Mice

Mice are selected with a C57BL/6 background which are back crossed intoC57BL/6. Offspring are genotyped using PCR using primers specific forwild-type or mutant bmf genes.

A bmf targeting vector is generated as shown in FIG. 5A. A neomycin orhygromycin sequence is used as the selectable marker. The construct isintroduced into embryonic stem cells and transformed cells selectedusing neomycin or hygromycin. The transformed embryonic stem cells arethen used to generate genetically modified mice.

EXAMPLE 6 Genomic Organization of Bmf and Identification of PromoterRegions

The genomic organization of the murine bmf gene is shown in FIG. 5A.Upstream of this region comprises a promoter region as outlined in SEQID NO:9. A corresponding promoter from the human bmf gene is outlined inSEQ ID NO:10.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. A nucleic acid molecule comprising a nucleotide sequence encoding orcomplementary to a sequence encoding an amino acid sequencesubstantially as set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof or having atleast about 45% or greater similarity to one or more of SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homologthereof.
 2. The nucleic acid molecule of claim 1 comprising a nucleotidesequence which encodes the amino acid sequence set forth in SEQ ID NO:2.3. The nucleic acid molecule of claim 1 comprising a nucleotide sequencewhich encodes the amino acid sequence set forth in SEQ ID NO:4.
 4. Thenucleic acid molecule of claim 1 comprising a nucleotide sequence whichencodes the amino acid sequence set forth in SEQ ID NO:6.
 5. The nucleicacid molecule of claim 1 comprising a nucleotide sequence which encodesthe amino acid sequence set forth in SEQ ID NO:8.
 6. The nucleic acidmolecule of claim 1 comprising a nucleotide sequence set forth in SEQ IDNO:1 or a nucleotide sequence having at least about 45% similaritythereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:1or its complementary form under low stringency conditions.
 7. Thenucleic acid molecule of claim 1 comprising a nucleotide sequence setforth in SEQ ID NO:3 or a nucleotide sequence having at least about 45%similarity thereto or a nucleotide sequence capable of hybridizing toSEQ ID NO:3 or its complementary form under low stringency conditions.8. The nucleic acid molecule of claim 1 comprising a nucleotide sequenceset forth in SEQ ID NO:5 or a nucleotide sequence having at least about45% similarity thereto or a nucleotide sequence capable of hybridizingto SEQ ID NO:5 or its complementary form under low stringencyconditions.
 9. The nucleic acid molecule of claim 1 comprising anucleotide sequence set forth in SEQ ID NO:7 or a nucleotide sequencehaving at least about 45% similarity thereto or a nucleotide sequencecapable of hybridizing to SEQ ID NO:7 or its complementary form underlow stringency conditions.
 10. The nucleic acid molecule of claim 1comprising the nucleotide sequence set forth in SEQ ID NO:1.
 11. Thenucleic acid molecule of claim 1 comprising the nucleotide sequence setforth in SEQ ID NO:3.
 12. The nucleic acid molecule of claim 1comprising the nucleotide sequence set forth in SEQ ID NO:5.
 13. Thenucleic acid molecule of claim 1 comprising the nucleotide sequence setforth in SEQ ID NO:7.
 14. An isolated protein comprising an amino acidsequence encoded by a nucleotide sequence set forth in SEQ ID NO:1 orSEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or a nucleotide sequencehaving at least about 45% similarity to the nucleotide sequence setforth in SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or anucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3or SEQ ID NO:5 or SEQ ID NO:7 or a complement thereof under lowstringency conditions.
 15. The isolated protein of claim 14 comprisingan amino acid sequence encoded by the nucleotide sequence set forth inSEQ ID NO:11.
 16. The isolated protein of claim 14 comprising an aminoacid sequence encoded by the nucleotide sequence set forth in SEQ IDNO:3.
 17. The isolated protein of claim 14 comprising an amino acidsequence encoded by the nucleotide sequence set forth in SEQ ID NO:5.18. The isolated protein of claim 14 comprising an amino acid sequenceencoded by the nucleotide sequence set forth in SEQ ID NO:7.
 19. Theisolated protein of claim 14 comprising an amino acid sequence as setforth in SEQ ID NO:2 or having at least 45% similarity thereto.
 20. Theisolated protein of claim 14 comprising an amino acid sequence as setforth in SEQ ID NO:4 or having at least 45% similarity thereto.
 21. Theisolated protein of claim 14 comprising an amino acid sequence as setforth in SEQ ID NO:6 or having at least 45% similarity thereto.
 22. Theisolated protein of claim 14 comprising an amino acid sequence as setforth in SEQ ID NO:8 or having at least 45% similarity thereto.
 23. Theisolated protein of claim 14 having an amino acid sequence as set forthin SEQ ID NO:2.
 24. The isolated protein of claim 14 having an aminoacid sequence as set forth in SEQ ID NO:4.
 25. The isolated protein ofclaim 14 having an amino acid sequence as set forth in SEQ ID NO:6. 26.The isolated protein of claim 14 having an amino acid sequence as setforth in SEQ ID NO:8.
 27. A variant of an isolated bmf nucleic acidmolecule comprising one or more nucleotide mutations in said nucleicacid molecule resulting in at least one amino acid addition,substitution and/or deletion to the polypeptide encoded by said variantwherein said polypeptide cannot bind, couple or otherwise associate witha dynein light chain, such as DLC2.
 28. The variant of claim 28 whereinthe mutation results in an altered amino acid sequence in the regionwhich binds to the dynein light chain.
 29. A variant of an isolated Bmfpolypeptide comprising at least one amino acid addition, substitutionand/or deletion wherein said polypeptide cannot bind, couple orotherwise associate with the dynein light chain.
 30. A method ofmodulating activity of Bmf in a mammal, said method comprisingadministering to said mammal a modulating effective amount of an agentfor a time and under conditions sufficient to increase or decrease Bmfactivity.
 31. A method of modulating apoptosis in a mammal, said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofa nucleotide sequence encoding bmf.
 32. A method of modulating apoptosisin a mammal, said method comprising administering to said mammal aneffective amount of an agent for a time and under conditions sufficientto modulate the activity of Bmf.
 33. A method of treating a mammal, saidmethod comprising administering to said mammal an effective amount of anagent for a time and under conditions sufficient to modulate theexpression of bmf wherein said modulation results in modulation ofapoptosis.
 34. A method of treating a mammal, said method comprisingadministering to said mammal an effective amount of an agent for a timeand under conditions sufficient to modulate the activity of Bmf whereinsaid modulation results in modulation of apoptosis.
 35. The method ofclaim 30 or 31 or 32 or 33 or 34 wherein the mammal is a human.
 36. Apharmaceutical composition comprising bmf, Bmf or derivative thereof oran agent capable of modulating bmf expression or Bmf activity togetherwith one or more pharmaceutically acceptable carriers and/or diluents,bmf, Bmf or said agent are referred to as the active ingredients.
 37. Amonoclonal antibody having specificity for Bmf or bmf or derivativethereof.
 38. A method of detecting an immunointeractive molecule, in asample, specific for a protein of interest produced by a cell, saidmethod comprising contacting the sample to be tested with a populationof cells comprising a defined ratio of cells producing the protein ofinterest and cells not producing the protein of interest for a time andunder conditions sufficient for immunointeractive molecules, if presentin said sample, to interact with said protein of interest and thesubjecting said immunointeractive molecule-protein complex to detectingmeans.
 39. The method of claim 38 wherein the interactive molecule is anantibody.
 40. A genetically modified animal in which one or both allelesof bmf are mutated alone or in combination with another mutation in oneor both alleles for another Bcl-2 molecule such as but not limited togenes encoding Blk, Bad, Bik, Hrk, Bid, Bim, Noxa and/or Puma.
 41. Thegenetically modified animal of claim 40 wherein said animal is a mouse.42. The genetically modified animal of claim 40 wherein said animal is arat.
 43. The genetically modified animal of claim 40 wherein said animalis a pig.
 44. A method of producing a genetically modified non-humananimal, said method comprising introducing into embryonic, stern cellsof an animal a genetic construct comprising a bmf nucleotide sequencecarrying a single or multiple nucleotide substitution, addition and/ordeletion or inversion or insertion wherein there is sufficient bmfnucleotide sequences to promote homologous recombination with a bmf genewithin the genome of said embryonic stem cells selecting for saidhomologous recombination and selecting embryonic stem cells which carrya mutated bmf gene and then generating a genetically modified animalfrom said embryonic stem cell.
 45. The method of claim 44 wherein thegenetically modified animal is a mouse or rat.
 46. Use of Bmf in themanufacture of a medicament for the treatment of a condition in a human.47. Use of Bmf in the manufacture of a medicament for the treatment of acondition in a non-human.